Page 44 - 南京医科大学学报自然科学版

南京医科大学学报（自然科学版） 第43卷第3期
·334 · Journal of Nanjing Medical University（Natural Sciences） 2023年3月

·基础研究·

佛手柑内酯对人牙髓干细胞成骨分化的影响

丁浩南 ，严 佳 ，唐诗佳 ，章非敏 1，2*
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南京医科大学口腔疾病研究江苏省重点实验室，南京医科大学附属口腔医院修复科，江苏 南京 210029
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［摘 要］ 目的：探究佛手柑内酯（bergapten，BP）对人牙髓干细胞（human dental pulp stem cell，hDPSC）成骨分化的影响。方
法：流式鉴定hDPSC的表面特异性抗原；使用CCK⁃8法检测BP的生物毒性与生物安全性；将hDPSC随机分为对照组与BP组，
BP组分别加入含7.5、15.0、30.0 μmol/L BP 的完全培养基，对照组加入含等体积二甲基亚砜溶液（dimethyl sulfoxide，DMSO）的
完全培养基，采用荧光显微镜观察各组细胞形态。使用BCIP/NBT碱性磷酸酶（alkaline phosphatase，ALP）显色及ALP定量检测
法检测BP对hDPSC早期成骨分化的影响；使用茜素红（alizarin red S，ARS）染色法及半定量分析法评估成骨分化晚期细胞外
基质矿化程度；利用实时荧光定量聚合酶链式反应（quantitative real⁃time PCR，qRT⁃PCR）检测 BP 对于 Runt 相关转录因子 2
（Runt⁃related transcription factor 2，RUNX2）、骨钙素（osteocalcin，OCN）、骨桥蛋白（osteopontin，OPN）、ALP等成骨标志基因表达
水平的影响；使用免疫印迹分析检测成骨分化蛋白指标的变化。结果：CCK⁃8实验结果表明7.5、15.0、30.0 μmol/L BP生物安全
性较好（P < 0.01）；与对照组相比，BP组细胞ALP染色更明显，15.0 μmol/L组ALP活性水平更高（P < 0.01）；与对照组相比，BP
组细胞外基质矿化水平更高，15.0 μmol/L BP处理后结节最多最深；与对照组相比，BP组成骨相关基因和蛋白的表达均明显增
多且 15.0 μmol/L 处理略优（P < 0.01）。结论：一定浓度的 BP 具有良好的生物安全性，且能对 hDPSC 的成骨分化产生促进作
用，15.0 μmol/L成骨诱导效果较优。
［关键词］ 佛手柑内酯；人牙髓间充质干细胞；成骨分化；骨组织工程
［中图分类号］ R780.2 ［文献标志码］ A ［文章编号］ 1007⁃4368（2023）03⁃334⁃09
doi：10.7655/NYDXBNS20230306

Effects of bergapten on osteogenic differentiation of human dental pulp stem cells
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DING Haonan ，YAN Jia ，TANG Shijia ，ZHANG Feimin 1，2*
1 Jiangsu Province Key Laboratory of Oral Diseases，Nanjing Medical University；Department of Prosthodontics，the
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Affiliated Stomatological Hospital of Nanjing Medical University，Nanjing 210029，China
［Abstract］ Objective：The current study aims to explore the effect of bergapten（BP）on the osteogenic differentiation of human
dental pulp stem cells（hDPSCs）. Methods：Specific antigens of hDPSCs were identified by flow cytometry analysis. The cytotoxicity
and biosafety of BP was detected by CCK ⁃ 8. The hDPSCs were randomly divided into the control and BP groups. Different
concentrations of BP（7.5 μmol/L，15.0 μmol/L and 30.0 μmol/L）were added to complete medium in the BP groups，while an equal
volume of DMSO was added to the complete medium in the control group. The morphology of hDPSCs in the BP and control groups was
observed by fluorescent stain. The BCIP/NBT staining and alkaline phosphatase（ALP）quantitative detection were used to detect the
effect of BP at the early stage of osteogenic differentiation of hDPSCs. The alizarin red S（ARS）staining and semi⁃quantitative analysis
was used to assess the degree of extracellular matrix mineralization at the late stage of osteogenic differentiation. Expressions of
osteogenic⁃related genes such as Runt⁃related transcription factor 2（RUNX2），osteocalcin（OCN），osteopontin（OPN），and ALP were
detected by quantitative real⁃time PCR（qRT⁃PCR）. Expressions of osteogenic⁃related proteins were detected using Western blot.
Results：BP of 7.5 μmol/L，15.0 μmol/L and 30.0 μmol/L showed better biosafety（P < 0.01）. The ALP activities in the BP groups
were higher than that in the control group，and the 15.0 μmol/L BP was the best（P < 0.01）. The degrees of extracellular matrix
mineralization of the BP groups were higher than that of the control group，and the number of calcium nodules in the 15.0 μmol/L BP
treatment group was more and deeper. The expressions of osteogenic⁃related genes and proteins were significantly increased in the BP

［基金项目］ 国家重点研发计划（2021YFA1201302，2021YFA1201300）；江苏省高校优势学科建设工程资助项目（2018⁃87）
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通信作者（Corresponding author），E⁃mail：fmzhang@njmu.edu.cn